survival of agrobacterium radiobacter k84 on various

Vol. 57, No. 7APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1991, p. 2047-20510099-2240/91/072047-05$02.00/0

Survival of Agrobacterium radiobacter K84 on VariousCarriers for Crown Gall Control

BEATRICE PESENTI-BARILI, ENRICA FERDANI, MARCO MOSTI, AND FRANCESCO DEGLI-INNOCENTI*Ricerca Biotecnologica per l'Agricoltura, Centro Ricerche, Agrimont S.p.A., via Massa Avenza 85,

I-54100 Massa, ItalyReceived 19 February 1991/Accepted 1 May 1991

Screening was performed on nine carriers to find an improved formulation for Agrobacterium radiobacter K84cells. The survival data showed that it is possible to preserve A. radiobacter cells on dry solid supports for a longtime provided that the storage temperature is 4°C and that the inoculation volume for 4 x 109 CFU g'- is notless than 0.15 ml g of carrier'1. On the other hand, a substantial carrier water content was necessary for roomtemperature storage. Many materials proved to be suitable as microbial carriers; in some cases, vermiculiteallowed long storage times comparable to those reported for peat or carboxymethyl cellulose, which are alreadyemployed in some commercial A. radiobacterK84 products. Furthermore, vermiculite assured full and immediatebiological activity in the prevention of crown gall, showing that it is suitable for a new formulation of strain K84.A hypothesis to explain the different survival abilities in wet and dry conditions is presented.

Crown gall, which is caused by the soilborne bacteriumAgrobacterium tumefaciens, is a serious disease affectingdicotyledonous plants in many parts of the world (18).Among the economically important hosts are members of thefamily Rosaceae (almond, apple, stone fruits, raspberry,blackberry, and rose), chrysanthemums, grape vines, pecantrees, walnut trees, and willows (4). The pathogen infectsthrough wounds frequently inflicted during transplantationand other agricultural operations. So far, chemical control ofthe disease has been mostly ineffective (7, 18). On the otherhand, the biological control of crown gall by the bacteriumAgrobacterium radiobacter K84 is effective and economic,and the strain is commercialized and used in agriculture. Asa matter of fact, the use of strain K84 is considered anoutstanding example of effective microbial biological control(17). Several reviews (8, 9, 11, 12) describe the intensivework done by many groups on the strain isolated by Newand Kerr (13).

Strain K84 is commercially supplied as a culture in agarplates (Galltroll; AgBioChem, Inc., Orinda, Calif.), in aformulation containing carboxymethyl cellulose (CMC)(Norbac 84-C; Nortell Laboratories, Inc., Corvallis, Oreg.),and in a finely ground peat preparation similar to Rhizobiuminoculum (several companies) (11). Furthermore, a methodof producing freeze-dried inocula has been proposed (3). Tobe successful, a microbial product, besides being effective,should have the requisites normally found in the traditionalsynthetic products: commercially acceptable storage time (atleast 1 year) at room temperature, simple use, and lowproduction costs.The agar plates present many drawbacks: short storage

time at low temperatures (maximum of 120 days at 4°C), theneed to manipulate an aesthetically unpleasant bacterialmass, and a laborious production process with high workercosts.The products based on CMC are reported to offer good

viability at low temperatures (a drop in viable count of afactor of <10 in 11 months at 4°C) (11). However, it seemsthat K84 cells stored in CMC, although viable, are retarded

* Corresponding author.

physiologically because colonies take about 5 days to appearon nutrient agar (11). Good results have been obtained withpeat; K84 can be stored in peat for at least 4 months at roomtemperature before its titer is reduced 10-fold (21). However,peat, being an organic material extracted from natural de-posits, shows great variability. Samples coming from dif-ferent deposits, as well as batches from the same source,affect microbial survival differently. Furthermore, peat pres-ents sterilization problems because it produces toxic sub-stances when treated with heat, gamma radiation, or gaseouschemical agents (6, 20).With the purpose of finding a suitable carrier for A.

radiobacter with improved features (acceptable storage timeat room temperature and substrates of constant quality thatare easy to sterilize), we determined whether preservingcells on dry solid supports was possible. Usually, dryformulations are used for dormant microbes, namely, spore-forming gram-positive bacteria (14). However, the use ofdehydrating carriers, such as anhydrous silica gel, is suc-cessfully applied for the preservation of filamentous fungi-not only conidia (dormant cells) but also mycelia, i.e.,fragile, vegetative cells (15). We therefore wanted to testwhether A. radiobacter cultures dehydrated without a freez-ing step (as in lyophilization) on dry supports could besuccessfully preserved. For this purpose, nine materialswere screened to select those with the best qualities.

MATERIALS AND METHODS

Bacterial strain. A. radiobacter K84 (NCPPB 2407) and apathogenic A. tumefaciens strain (NCPPB 1651) were sup-plied by the National Collection of Plant Pathogenic Bacte-ria, Harpenden, United Kingdom. The pathogenicity of A.tumefaciens was assessed by tests on carrot discs (1). Thesensitivity of the A. tumefaciens strain to agrocin 84 wasverified by the Stonier method (19).Media. The media used for growing A. radiobacter K84

were nutrient broth medium and YDPC medium. YDPCmedium contained the following ingredients (per liter): yeastextract, 4 g; peptone, 4 g; (NH4)2SO4, 5 g; CaCO3, 10 g; andglucose, 20 g. The final pH was 7. Glucose was added as aseparately sterilized solution. Nutrient broth medium, pep-

2047

2048 PESENTI-BARILI ET AL.

tone, and yeast extract were from Difco (Detroit, Mich.); allof the other reagents were from Farmitalia Carlo Erba(Milan, Italy).Growth conditions. The strain was precultured in nutrient

broth medium for 1 day at 24°C. Next, a 25-ml aliquot of thisculture was transferred into 800 ml of YDPC medium in a2-liter Erlenmeyer flask and cultured on a rotary shaker at250 rpm for 2 days at 24°C. The culture was harvested bycentrifugation and resuspended with protective solution.

Carrier materials. Nine materials were screened as carri-ers for A. radiobacter: kaolin (Caolino Summano; Posina,Vicenza, Italy); silica gel (Carlo Erba); vermiculite (expand-ed type) and agriperlite (Vic Italiana, Milan, Italy); expandedclay (Ares 4; Trino, Vercelli, Italy); and four types ofdiatomaceous products, Celite and Micro-cel (Johns-Man-ville Products Co., Lompoc, Calif.), Diatom (Europe SudS.p.A., Milan-Corsico, Italy), and Porosil MP (Ceca, Milan,Italy). The materials were washed (except for the powders[kaolin, Celite, Porosil MP, Micro-cel, and Diatom]) withdistilled water and dried by heating at 80°C; the weightvariation was monitored with a precision balance. Thematerials were considered dry when no further weight losswas detectable. The carriers (3 g) were dispensed intoscrew-cap borosilicate glass tubes (16 by 150 mm; CarloErba). The tubes were then autoclaved at 121°C for 1 h anddried by heating at 150°C until their weights were constant.In a second experiment, the carriers were sterilized byautoclaving twice at 121°C for 1 h, with an interval of 24 hafter the first treatment, dried by heating at 80°C until aconstant weight was reached, and aseptically dispensed (10g) into polyethylene bags (bag size, 100 by 150 mm; CarloErba), which are commercially more suitable containersthan glass tubes.The carrier water content was determined as the differ-

ence between the weight of wet material and the weight ofdry material.

Preparation of inoculant. A 5% (wt/vol) sucrose solution insterile skim milk was used as a protective solution. Eachglass tube was inoculated with 0.5 ml of the bacterialsuspension, shaken thoroughly to mix its contents, andstored at 4°C or 21°C. The final moisture content of each tubewas about 14% (wt/wt). In a second experiment, the culturewas resuspended with a solution of 10% (wt/vol) skim milkpowder (Mag-ist; Gespal, Milan, Italy) and 7% (wt/vol)sucrose. Two bacterial suspensions were prepared, with onethree times more concentrated than the other. The carriers,contained in plastic bags, were inoculated either with 0.5-mlaliquots of the concentrated suspension (moisture, 4.7%) orwith 1.5-ml aliquots of the diluted suspension (moisture,13%). The bags were then heat sealed and stored at 4 or240C.

Viable-cell counts. To determine the survival rate of thebacteria, 20 ml of normal saline (0.88% [wt/vol] NaCi) wasadded to each sample. The suspension was shaken for 2 minwith a wrist-action shaker, and then serial dilutions werespread on nutrient agar plates. The plates were incubated at240C and scored after 2 to 3 days. Colonies appearing in lessthan 2 days were not agrobacteria and thus were noted ascontaminants. The number of CFU in the suspension wasexpressed as CFU per gram of carrier.

Biological effectiveness of inoculants. The inoculants weretested for effectiveness in the prevention of gall formation on3-week-old tomato plants (Lycopersicum esculentum Mill.cv. S. Pierre). The method was similar to that described byDu Plessis et al. (5). The inoculum of the pathogen wasprepared by washing cells from nutrient agar plates with

normal saline, counting the cells, and diluting the suspensionto the desired concentration (about 4 x 108 cells ml-'). AnA. radiobacter suspension was drawn from the carrier asdescribed above for viable counts. The two suspensionswere mixed in different ratios, and 25-pl aliquots of thesemixtures were injected into stems at the first internode witha sterile 1-ml syringe. At least five plants were used for eachdetermination. Tumor production was scored 4 weeks afterinoculation. The gall size was determined by subtracting thestem diameter of a healthy plant from the stem diameter of aplant with a gall.

Statistical analysis. To compare the different formulations,degradation rates (regression coefficients) were calculatedby regression analysis using the simple regression procedureof Statgraphics software (STSC, Inc., Rockville, Md.), withthe base 10 logarithm of the cell concentration (in CFU pergram) as the dependent variable and the time (in days) as theindependent variable.The 95% confidence limits were calculated by multiplying

the regression coefficient's standard error by Student's tvalue. R2 (coefficient of determination) measures the contri-bution of the linear function of the independent variables tothe variation in the dependent variable.

RESULTS

A. radiobacter K84 cells were grown with nine differentcarriers. The data for silica gel are not reported because thismaterial was very toxic to A. radiobacter K84. The survivalof the bacteria was monitored by determining the number ofCFU per gram of carrier at different times after inoculation.Two independent samples were assayed for each determina-tion. Some samples (10 of 208) were lightly contaminatedand thus were discarded. The contaminated samples werefound more frequently at 21°C (7 of 96 samples) than at 4°C(3 of 112) and more frequently in granular materials, such asagriperlite (3 of 26), expanded clay (2 of 26), and vermiculite(2 of 26), than in powdered carriers (O or 1 of 26 samples foreach material). This could be a result of a better heatexchange in the powdered carriers. However, the powderedcarriers were difficult to wash because of the formation ofthick suspensions. The results are shown in Table 1 as thebase 10 logarithm of the mean number of CFU per gram. Theregression coefficients with the 95% probability confidencelimits, the R2 values, and the maximum storage times for thecarriers stored at 21 and 4°C are shown in Tables 2 and 3,respectively. The maximum storage time is the number ofdays needed to have a drop in the viable-cell count of afactor of 100 and is calculated as -loglOO x regressioncoefficient-1. The viability decrease in the storage timedefinition (100 times) is not an arbitrary value, but it delimitsthe minimum acceptable titer, considering that (i) the initialcell concentration in our formulations is about 4 x 109 cellsg-1, (ii) the final cell concentration in the solution at themoment of inoculation of plants has to be at least 106 cellsml-1 (13), and (iii) the treatment solution is obtained bydiluting a gram of bacterial formulation in 30 to 40 ml ofwater. Therefore, the storage time is a real expiration timethat represents the threshold after which the vital cellconcentration is too low for effective K84 protection.The regression coefficients at 4°C (Table 3) were lower for

each carrier than the corresponding values at 21°C (Table 2),indicating better survival at a low temperature, regardless ofthe carrier used in the formulation.At 21°C, the differences between most carriers were small;

Porosil MP, expanded clay, and kaolin showed the best

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A. RADIOBACTER K84 SURVIVAL ON VARIOUS SUBSTRATES 2049

TABLE 1. Survival of A. radiobacter K84 in different carriers at 4 and 21°C

Carrier Temp Survival (log10 mean viable titer [CFU g-1]) at indicated time (days)( C) 0 1 45 80 175 244 314 393

Agriperlite 21 9.59 9.78 7.68 6.96 4.46a 1.78a NDb NDExpanded clay 21 9.15 9.23 7.40a 6.81a 6.66 4.39 ND NDKaolin 21 8.72 8.11 6.56 5.48 3.89 3.74 ND NDCelite 21 9.30 9.31 8.79a 6.79 3.89 3.40 ND NDDiatom 21 9.16 8.19 6.77 6.80 3.05 3.23 ND NDPorosil MP 21 9.29 9.56 9.10 7.22 5.95 6.30 ND NDMicro-cel 21 9.54 8.46 3.84 1.73 1.73a 1.73 ND NDVermiculite 21 9.33 9.33 8.81 7.97 6.11a 1.52 ND ND

Agriperlite 4 9.59 9.78 9.80 9.68 8.21 ND 6.20a 6.59Expanded clay 4 9.15 9.23 8.89 9.20 8.05 ND 7.08 4.65Kaolin 4 8.72 8.11 7.77 7.64 5.55 ND 5.41 3.91Celite 4 9.30 9.31 9.24 8.91 8.38 ND 7.29 6.72Diatom 4 9.16 8.19 8.07 7.47 7.24 ND 6.25 4.91Porosil MP 4 9.29 9.56 9.64 9.26a 8.78 ND 7.42 6.43Micro-cel 4 9.54 8.46 8.46 8.11 5.16 ND 1.73 2.13Vermiculite 4 9.33 9.33 9.46a 9.37 9.35 ND 8.71 7.70

a Single sample.b ND, not determined.

results, with storage times of greater than 3 months (Table 2).At 4°C, vermiculite provided the longest storage time (563days) and Micro-cel provided the shortest (99 days) (Table 3).Good carriers at 4°C, such as vermiculite and Celite, were

poor substrates at 21°C; likewise, carriers with good viabilityat 21°C, such as Porosil MP and expanded clay, did not excelat 4°C. This low correlation suggests that accelerated stabil-ity methods are not easily applicable in screening experi-ments. In those procedures, degradation rates at a number ofelevated temperatures are plotted according to the Arrheniusequation, and the resulting lines are extrapolated to giveestimated values at lower temperatures (2).The biological effectiveness of K84 cells stored in vermic-

ulite at 4°C for 314 days was tested with tomato plantscoinoculated with A. radiobacter suspensions (obtainedfrom vermiculite samples) and fresh pathogen cultures. Thegall size (mean ± standard deviation) was 8.02 ± 0.93 and7.10 ± 0.48 mm when the plants were inoculated with 1.7 x108 and 1.7 x 107 cells ofA. tumefaciens ml-. On the otherhand, gall formation was completely prevented when patho-gen-A. radiobacter mixtures were inoculated not only at a1:10 ratio but also at a 1:1 ratio, the minimum reported to beeffective (13).We wanted to confirm the results of the first experiment by

TABLE 2. Regression analysis of survival data for strain K84stored at 21°C on different carriers

Regression MaximumCarrier coefficienta R2 storage time

+ C.L., 10-2 (days)

Micro-cel -9.479 ± 5.223 A 96.8 21Agriperlite -3.115 + 0.425 B 99.0 64Vermiculite -2.963 ± 1.145 BC 92.8 67Celite -2.667 ± 0.783 BC 95.7 75Diatom -2.445 ± 1.049 BC 91.3 81Kaolin -1.997 ± 0.965 BC 89.2 100Expanded clay -1.711 ± 0.862 C 88.4 116Porosil MP -1.491 ± 0.893 C 84.3 133

a Values, shown with the 95% confidence limits (C.L.), followed by thesame letter do not differ significantly.

retesting Porosil MP and expanded clay (the two bestcarriers at 21°C) and vermiculite (the best carrier at 4°C) in asecond experiment. The higher storage temperature in thiscase was, for practical reasons, 24°C. In this experiment nosample was contaminated, probably as a consequence of thedifferent sterilization procedure (see Materials and Meth-ods).To simplify a possibly large-scale production process, we

determined whether it was possible to decrease the inoculumvolume while keeping the total number of bacteria constant.Some samples (denominated H for high inoculation volume)were inoculated with 1.5 ml of bacterial suspension (inocu-lum/carrier ratio, = 0.15 ml g'-, approximately equal to thatin the first experiment) and others (L for low inoculationvolume) were inoculated with 0.5 ml (inoculum/carrier ratio,= 0.05 ml g-1) of a threefold-more-concentrated suspension,so as to maintain a constant total number of bacteriaintroduced into each sample.We also wanted to test the effect of the carrier water

content on A. radiobacter survival. Bacteria were inoculatedas described above into dry and wet (water content = 75%[wt/wt]) vermiculite samples.The survival data are reported in Table 4 as the base 10

logarithm of the number of CFU per gram. In Table 5 and


Regression coefficienta 2 MaximumCarrier ± C.L., 10-2 (days)

Micro-cel -2.011 ± 0.535 A 94.9 99Kaolin -1.094 + 0.336 B 93.3 182Expanded clay -1.017 ± 0.414 BC 88.8 196Agriperlite -0.966 ± 0.329 BC 91.9 207Diatom -0.856 + 0.287 BCD 92.1 233Porosil MP -0.758 ± 0.223 BCD 93.8 263Celite -0.669 ± 0.075 C 99.0 299Vermiculite -0.355 ± 0.227 D 76.2 563

a Values, shown with the 95% confidence limits (C.L.), followed by thesame letter do not differ significantly.

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2050 PESENTI-BARILI ET AL.

TABLE 4. Survival of A. radiobacter K84 in different carriers at4 and 24°C

Survival (loglo) viable titer

Temp Inocu- [CFU g-1]) at indicatedCarrier (OC) lum' ~ time (days)

0 4 56 120 200

Expanded clay 24 H 9.68 9.53 7.34 4.00 2.34Expanded clay 24 L 9.81 9.70 5.60 5.28 2.88Porosil MP 24 H 9.68 9.45 7.00 2.00 1.00Porosil MP 24 L 9.81 9.48 4.00 1.30 1.00Vermiculite (wet) 24 H 9.68 9.60 8.51 8.30 8.04Vermiculite (wet) 24 L 9.81 9.82 8.20 8.08 7.83Vermiculite (dry) 24 H 9.12 8.58 8.08 2.70 1.00Vermiculite (dry) 24 L 9.24 8.75 7.15 2.70 1.00

Expanded clay 4 H 9.68 9.53 9.53 9.53 9.34Expanded clay 4 L 9.81 9.70 9.60 8.53 6.60Porosil MP 4 H 9.68 9.45 9.00 8.86 9.11Porosil MP 4 L 9.81 9.48 7.88 5.89 4.38Vermiculite (wet) 4 H 9.68 9.60 9.66 9.45 9.41Vermiculite (wet) 4 L 9.81 9.82 9.76 9.08 8.64Vermiculite (dry) 4 H 9.12 8.58 8.66 8.83 8.67Vermiculite (dry) 4 L 9.24 8.75 8.67 8.67 8.54

a H, high inoculation volume; L, low inoculation volume.

Table 6, the regression data for the carriers stored at 24 and4°C, respectively, are shown. Some carriers show a low R2because the datum points are scattered near the initial titer.The regression curve has a very low slope (roughly parallelto the x axis), and therefore the y values are poorly deter-mined by a linear function with the x values.The data confirm the properties of the carriers under study

that, when inoculated with the appropriate inoculation vol-ume (H samples) and stored at 4°C (Table 6), show evenbetter results than those of the first experiment (Table 3).The decay rates at 24°C (Table 5) were higher than those at21°C (Table 2), probably because of the higher storagetemperature used in the second experiment.The results also indicate that a threefold reduction of the

inoculum volume, when the total number of vital bacteriawas kept constant, did not cause any significant difference inthe degradation rate at 24°C (Table 5). However, inoculationwith a smaller volume detrimentally affected survival in allcarriers when they were stored at 4°C (differences betweenH and L samples were significant except for dry vermicu-


MaximumCarrier Inocu- Regression coeffi- R2 storageluma cientb C.L., 10-2 time

(days)

Expanded clay H -3.845 ± 1.1536 A 97.4 52Expanded clay L -3.370 ± 2.1410 AB 89.3 59Porosil MP H -4.687 ± 2.2447 A 93.6 42Porosil MP L -4.627 ± 3.8208 AB 83.2 43Vermiculite (wet) H -0.819 ± 0.6839 B 82.9 244Vermiculite (wet) L -1.012 ± 1.0455 B 76.0 197Vermiculite (dry) H -4.288 ± 2.1177 A 93.3 46Vermiculite (dry) L -4.298 ± 1.5441 A 96.3 46

a H, high inoculation volume; L, low inoculation volume.b Values, shown with the 95% confidence limits (C.L.), followed by the

same letter do not differ significantly.


Maximum

Carrier Inocu- Regression coefficient' R2 storageluma t C.L., 10-2 time(days)

Expanded clay H -0.121 ± 0.1356 A 73.1 1,647Expanded clay L -1.542 ± 0.8043 CD 92.5 130Porosil MP H -0.267 ± 0.5420 ABD 45.0 753Porosil MP L -2.732 + 0.6131 C 98.5 73Vermiculite (wet) H -0.130 + 0.1173 A 80.7 1,540Vermiculite (wet) L -0.617 ± 0.2655 BD 94.8 324Vermiculite (dry) H -0.071 ± 0.4465 AB 7.9 2,835Vermiculite (dry) L -0.227 ± 0.4140 AB 50.4 885

a H, high inoculation volume; L, low inoculation volume.b Values, shown with the 95% confidence limits (C.L.), followed by the

same letter do not differ significantly.

lite). Therefore, the inoculation volume is an importantfactor for survival at 4°C.The data obtained in the study of carrier water content

show that, at 4°C, bacteria inoculated into wet vermiculitehad decay rates slightly higher than those of bacteria inocu-lated into dry vermiculite (Table 6). On the other hand, at24°C the high water content remarkably and significantlyimproved the survival of strain K84 (Table 5).The biological activity of a wet-vermiculite H sample

stored at 24°C for 294 days was assayed with tomato plantsas described above. The titer of this sample was 2.5 x 107CFU g-1. The control gall size (mean + standard deviation)was 6.37 + 0.73 mm (with an inoculum of 1.9 x 107 cells ofA. tumefaciens ml-'). Gall formation was completely prc-vented at a 1:1 A. tumefacienslA. radiobacter ratio.

DISCUSSION

This work is part of a study which has been undertaken tofind a microbial formulation suitable for the bacterium A.radiobacter K84, which is used in agriculture for the preven-tion of crown gall. The aim is to define a product of constantquality, long storage time at room temperature, simple use,and low production costs. Some of these features are real-ized in already commercialized products, but not together inone product, with each formulation having some drawback.In this work, we wanted in particular to determine thefeasibility of preserving A. radiobacter cells by dehydrationon dry solid supports, a method used with filamentous fungi.For this purpose, nine materials were screened for bacterialsurvival for at least 200 days. The toxic effect of silica gelwas probably due to contamination with a few dye particles,which are not suitable for preservation purposes (15). Ver-miculite was one of the best carriers for A. radiobacter K84at 4°C. The vermiculite storage time is estimated to be verylong, on the order of years. The storage time values areextrapolations and therefore are to be judged with greatcaution. Nevertheless, considering the experimental datainstead of the regression estimates, we find that the viable-cell counts after 200 days are still about 40% of the initial cellconcentration, i.e., 40 times higher than the expiration limit(1%). Therefore, vermiculite offers, at 4°C, viability levelscomparable to those reported for CMC at the same temper-ature (11). Furthermore, cells maintained on vermiculite arenot as physiologically retarded as those maintained on CMC(11) but show full and immediate biological activity in theprevention of crown gall. At room temperature, the bacterial

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A. RADIOBACTER K84 SURVIVAL ON VARIOUS SUBSTRATES 2051

survival rate in dry carriers was generally low. However,good results, similar to those reported for peat (4 months ofstorage at room temperature with a 10-fold cell concentra-tion reduction) (21), were obtained with wet vermiculiteinstead of dry vermiculite, with a viable-cell count reductionof 100-fold in 6 to 7 months (Table 4). Compared with peat,vermiculite has the advantage of being sterilizable withouttoxicity problems.Our study of Agrobacterium formulation confirms the

useful characteristics of vermiculite as a microbial carrier,which have already been shown for Rhizobium strains andother bacterial species by Graham-Weiss et al. (6). Graham-Weiss et al. used vermiculite as a support medium for thedirect fermentation of bacterial cultures for the production ofreliable bacterial inoculants. Vermiculite is believed to allowa high survival rate by buffering pH shifts, adsorbing sup-pressive metabolites, and physically sheltering organismsagainst harsh environmental stress (22).The vermiculite water content has opposite effects on cell

viability at high and low storage temperatures. At 4°C thedegradation rates are slightly lower with dry vermiculite thanwith wet vermiculite, while at 24°C wet vermiculite yieldsbetter results. These findings indicate that the mechanismsof survival in the carrier in the presence and in the absenceof water are probably different. Perhaps, in the presence ofwater, bacteria are physiologically still active and starved;under these conditions, the low temperature represents afurther stress which is not well tolerated. It is known thatprotein synthesis during starvation is needed for survival viathe development of resistant cells of Escherichia coli. E. colicells starved for 5 h at 37°C survived much better whentransferred to 4°C than cells starved only for 10 min,indicating that the immediate exposure to a low temperatureimpaired starvation protein synthesis (10). On the otherhand, the low water availability may block or slow down thebiochemical pathways, allowing a high level of bacterialsurvival; however, under these conditions some importantcell component becomes particularly unstable and needs alow temperature to maintain its functions. Structural injury,defined as a transient and reversible susceptibility to agentsthat are innocuous in unstressed populations, has beenobserved in dried microorganisms (16). From these reports,we can say that it is probable that physiologically active A.radiobacter cells need to synthesize starvation proteinsbefore exposure to a temperature of 4°C and that dried A.radiobacter cells have a structural injury which causessensitivity to a temperature of 24°C.Our data showed that the inoculation volume is an impor-

tant factor for survival at 4°C but is negligible at 24°C.Decreasing the inoculation ratio from 0.15 to 0.05 ml g-1while maintaining a constant total number of bacteria re-sulted in a dramatic decrease in cell viability at 4°C. In thisexperiment, by lowering the inoculum volume, we changedtwo variables, namely, the amount of milk-sucrose solutionintroduced into each sample and the bacterial concentrationin the inoculum suspension. Therefore, the decrease inviability could be due to a detrimental effect of a too-high cellconcentration or to a smaller amount of protective solution.Other experiments will be necessary to clarify which are theimportant parameters and to determine their optimum levels.

ACKNOWLEDGMENTS

We thank Carlo Minganti for advice and encouragement, EnricoSelva and Paolo Angelini for reading the manuscript, and GiorgioBattolla for plant cultivation.

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